1. Field of Art
The present invention relates to the biochemical arts, particularly to recombinant expression of polypeptides.
2. Discussion of Related Art
Bioactive or therapeutic peptides can be potent drugs which specifically target and modulate unique signaling and metabolic pathways. Their relatively small size and simple composition makes these peptides amenable to molecular engineering to refine and enhance desirable activities. Subtle changes to the peptide sequence can discriminate between linked activities or help prevent degradation in vivo. Similarly, well placed linker sites can permit conjugation of large molecules, such as poly(ethylene glycol) PEG, to enhance circulating half-lives. However, these same properties also present special challenges to peptide production and delivery.
Artificial synthetic techniques are not cost-effective for producing many peptides, particularly the larger peptides (15-40 amino acid residues or more). As an alternative, the use of recombinant host cells is well known for recombinant production of bioactive peptides or proteins. Commonly used recombinant host cells include bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insect cells, plant cells, and mammalian cells in culture. However, recombinant expression is often difficult. One reason for the low expression of recombinant peptides or proteins is likely due to their poor refolding potential, owing to marginally stable secondary and tertiary structures in solution.
To overcome this, many peptides have been expressed as chimeric fusions with proteins such as immunoglobulin Fc domains, ubiquitin, an albumin (e.g., human serum albumin (HSA)), a transthyretin (TTR), or a thyroxine-binding globulin (TBG). (See, e.g., Sullivan et al., Toxin Peptide therapeutic agents, WO 2006/116156 A2; Gegg et al., Modified Fc molecules, WO 2006/036834 A2; Gegg et al., Modified Fc molecules, PCT/US2006/031609; Feige et al., Modified peptides as therapeutic agents, WO 2000/024782; Rosen et al., Albumin fusion proteins, U.S. Pat. No. 6,926,898 and US 2005/0054051; Bridon et al., Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components, U.S. Pat. No. 6,887,470); Walker et al., Use of transthyretin peptide/protein fusions to increase the serum half-life of pharmacologically active peptides/proteins, US 2003/0195154 A1; 2003/0191056 A1). Such large fusion proteins have made possible the commercial expression of therapeutic peptides and provided the added advantage of dramatically extending the circulating half-lives of their peptide partners, thereby rendering them more efficacious in vivo.
While these fusion proteins often facilitate peptide expression at much higher levels, they can also present difficult refolding challenges that can affect their bioactivity. Protein recovery can be further complicated by undesirable domain-domain interactions between the fusion partners and disulphide bond isomerizations. In addition, the cost of producing a fusion protein with a large protein carrier moiety can affect the commercial viability of such a therapeutic agent.
Consequently, compositions and methods for high yield recombinant expression of bioactive fusion proteins with a relatively low mass ratio of carrier component to bioactive component are desirable. These and other benefits are provided by the present invention.